Supercritical HPLC

TABLE 23.1 Potential mobile phases for supercritical HPLC... 

Mixtures can be identified with the help of computer software that subtracts the spectra of pure compounds from that of the sample. For complex mixtures, fractionation may be needed as part of the analysis. Commercial instmments are available that combine ftir, as a detector, with a separation technique such as gas chromatography (gc), high performance Hquid chromatography (hplc), or supercritical fluid chromatography (96,97). Instmments such as gc/ftir are often termed hyphenated instmments (98). Pyrolyzer (99) and thermogravimetric analysis (tga) instmmentation can also be combined with ftir for monitoring pyrolysis and oxidation processes (100) (see Analytical methods, hyphenated instruments). 

The development of methods of analysis of tria2ines and thek hydroxy metabohtes in humic soil samples with combined chromatographic and ms techniques has been described (78). A two-way approach was used for separating interfering humic substances and for performing stmctural elucidation of the herbicide traces. Humic samples were extracted by supercritical fluid extraction and analy2ed by both hplc/particle beam ms and a new ms/ms method. The new ms /ms unit was of the tandem sector field-time-of-flight/ms type. 

Supercritical Fluid Chromatography. Supercritical fluid chromatography (sfc) combines the advantages of gc and hplc in that it allows the use of gc-type detectors when supercritical fluids are used instead of the solvents normally used in hplc. Carbon dioxide, -petane, and ammonia are common supercritical fluids (qv). For example, carbon dioxide (qv) employed at 7.38 MPa (72.9 atm) and 31.3°C has a density of 448 g/mL. 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

Supercritical fluid chromatography (SFC) refers to the use of mobile phases at temperatures and pressures above the critical point (supercritical) or just below (sub-critical). SFC shows several features that can be advantageous for its application to large-scale separations [132-135]. One of the most interesting properties of this technique is the low viscosity of the solvents used that, combined with high diffusion coefficients for solutes, leads to a higher efficiency and a shorter analysis time than in HPLC. 

Purification of poloxamers has been extensively investigated due to their use in medical applications, the intention often being to remove potentially toxic components. Supercritical fluid fractionation and liquid fractionation have been used successfully to remove low-molecular weight impurities and antioxidants from poloxamers. Gel filtration, high-performance liquid chromatography (HPLC), and ultrafiltration through membranes are among the other techniques examined [5]. 

A number of analytical techniques such as FTIR spectroscopy,65-66 13C NMR,67,68 solid-state 13 C NMR,69 GPC or size exclusion chromatography (SEC),67-72 HPLC,73 mass spectrometric analysis,74 differential scanning calorimetry (DSC),67 75 76 and dynamic mechanical analysis (DMA)77 78 have been utilized to characterize resole syntheses and crosslinking reactions. Packed-column supercritical fluid chromatography with a negative-ion atmospheric pressure chemical ionization mass spectrometric detector has also been used to separate and characterize resoles resins.79 This section provides some examples of how these techniques are used in practical applications. 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

Teng, W.Y., Chen, C.C., and Chung, R.S., HPLC comparison of supercritical fluid extraction and solvent extraction of coumarins from the peel of Citrus maxima fmit, Phytochem. Anal, 16, 459, 2005. 

If simple sample pretreatment procedures are insufficient to simplify the complex matrix often observed in process mixtures, multidimensional chromatography may be required. Manual fraction collection from one separation mode and re-injection into a second mode are impractical, so automatic collection and reinjection techniques are preferred. For example, a programmed temperature vaporizer has been used to transfer fractions of sterols such as cholesterol and stigmasterol from a reversed phase HPLC system to a gas chromatographic system.11 Interfacing gel permeation HPLC and supercritical fluid chromatography is useful for nonvolatile or thermally unstable analytes and was demonstrated to be extremely useful for separation of compounds such as pentaerythritol tetrastearate and a C36 hydrocarbon standard.12... 

M. Yoshioka, S. Parvez, T. Miyazaki and H. Parvez, Supercritical Fluid Chromatography and Micro HPLC, VSP, Utrecht (1989). 

The nature of a supercritical fluid enables both gas and liquid chromatographic detectors to be used in SFC. Flame ionization (FID), nitrogen phosphorus (NPD), flame photometric (FPD) GC detectors (p. 100 etseq.) and UV and fluorescence HPLC monitors are all compatible with a supercritical fluid mobile phase and can be adapted to operate at the required pressures (up to several hundred bar). A very wide range of solute types can therefore be detected in SFC. In addition the coupled or hyphenated techniques of SFC-MS and SFC-FT-IR are attractive possibilities (cf. GC-MS and GC-IR, p. 114 el seq.). 

In supercritical fluid chromatography (SFC) the mobile phase is a supercritical fluid, such as carbon dioxide [15]. A supercritical fluid can be created either by heating a gas above its critical temperature or compressing a liquid above its critical pressure. Generally, an SFC system typically has chromatographic equipment similar to a HPLC, but uses GC columns. Both GC and LC detectors are used, thus allowing analysis of samples that cannot be vaporized for analysis by GC, yet cannot be detected with the usual LC detectors, to be both separated and detected using SFC. SFC is also in other... 

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